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The goal of this project is to characterize DNA damage by the epihalohydrins. In the past year, we have made progress in the following areas:

1. We are exploring the structure-function relationship of cross-linking by comparing epichlorohydrin (ECH) cross-linking to that of (1-chloroethenyl) oxirane.
2. We are characterizing the mechanism of cell death induced by ECH and (1-chloroethenyl) oxirane in 6C2 chicken erythroid cells.

Goal 1 Accomplishments      We are investigating the structure-function relationship for cross-linking. b-Chloroprene is an important industrial chemical used in manufacture of polychloroprene, a solvent-resistant elastomer. Correlation between occupational exposure to chloroprene and lung cancer has been reported. The active compound is believed to be (1-chloroethenyl)oxirane (CEO), a bifunctional metabolite structurally similar to ECH. We have synthesized this compound and found evidence for DNA interstrand cross-linking. Our data suggest cross-linking at deoxyguanosine residues within 5’-GC and 5’-GGC sites, with the rate of cross-linking depending on pH (pH 5.0 > pH 6.0 > pH 7.0). A comparison of the cross-linking efficiencies of CEO and the structurally similar cross-linkers diepoxybutane (DEB) and ECH revealed that DEB > CEO ≥ ECH. Furthermore, we have found that the relative cytotoxicites of these compounds in chicken 6C2 erythroid cells follows the same order, suggesting that interstrand cross-links are a primary contributor to cell death.

Goal 2 Accomplishments      We are exploring the role of apoptosis (programmed cell death) versus necrosis (direct damage) in the cytotoxicity of ECH and related compounds. Bifunctional alkylating agents react with many biological molecules in vivo. Reaction with DNA halts the cell cycle for DNA repair, which can trigger apoptosis. However, many cancer cells have defects in apoptotic control, in which case necrosis can be the mechanism of cytotoxicity.      We have successfully used two different assays for apoptosis within 6C2 chicken cells. The first monitors activity of the apoptosis-signaling proteins caspase-3/7 via cleavage of a pro-luminescent substrate. The second uses flow cytometry with Annexin V-FITC/Propidium Iodide (PI) staining. A fluorescent signal from Annexin V-FITC indicates phosphatidylserine exposure, which, coupled with exclusion of the plasma-membrane integrity probe PI, is suggestive of apoptosis. Our results suggest that with a 24-h treatment, ECH and CEO are weakly apopotic in comparison to DEB, with DEB >> ECH > CEO.  This suggests that interstrand cross-linking alone does not account for the apoptotic potential of these compounds.

Impact on Students      The Department of Chemistry at Colby College is firmly committed to providing significant independent research opportunities for our students, and we therefore require independent research of all our majors. Independent research reinforces classroom instruction and requires students to develop time-management skills, perseverance, and the ability to work in a group setting. Several students have been involved in this work in the Millard laboratory over the past year. Rebecca Kamins ’09 is attending medical school at Loyola University, Brian Wadugu ’09 is working as a research assistant at Boston Children’s Hospital, Bethany Bartley ’10 is entering her senior year at Colby College, and Christopher Ng ’10 is enrolled in the dual-degree engineering program at Colby College, spending his junior year at the Thayer School of Engineering at Dartmouth College. Ms. Kamins and Mr. Wadugu both presented their work at the 100^th Annual Meeting of the American Association for Cancer Research in Denver CO this spring, and Ms. Bartley and Mr. Ng both presented their work at the Colby Undergraduate Summer Research Retreat this summer.